1. Field of the Invention
The invention relates to a method of diagnosing tuberculosis by culturing lymphocytes from a subject to produce antibodies, and measuring the concentration of antibodies reactive with a tuberculosis antigen.
2. Background Information
Tuberculosis (TB) remains a major global health problem and is the most frequent cause of death from a single infectious agent [1]. The appearance of multidrug-resistant strains of Mycobacterium tuberculosis and the HIV/AIDS epidemic have contributed to the resurgence of active TB in humans. Thus, WHO declared tuberculosis a global emergency in 1993. Surveys carried out in Bangladesh from 1987 to the present suggest the smear positive TB case rate in Bangladesh to be between 1-1.8% [2-5].
Early diagnosis of TB is crucial to prevent the spread of the disease in the community. However, the clinical and laboratory diagnosis, follow-up of the infection activity and response to the therapy is not always easy to evaluate [6, 7]. Although, culture of bacteria is the gold standard in diagnosis and follow-up of disease, it can take up to 6-8 weeks to isolate M. tuberculosis. It is estimated that a false negative culture result may be obtained in 10-20% of TB cases [8, 9]. A rapid serological test for diagnosis, follow-up of disease activity and response to therapy would be very useful to the clinicians [10, 11]. The PPD skin test (Mantoux test) is an important tool for diagnosis of latent TB infection and disease in the developed world but it has low predictive value in Bacillus-Calmette-Guerin (BCG)-vaccinated individuals as well as in individuals living in areas endemic for TB due to cross-reactivity with BCG and atypical mycobacteria, and false negative reactions in malnourished children [12-14].
BCG has been used as an antigen in enzyme immunoassays in in vitro studies to determine the disease activity but was aborted due to difficulties in interpretation, or differentiating between active or past disease, and low sensitivity and specificity, respectively [15-19]. With the identification of regions of M. tuberculosis genome that are missing in BCG and nontuberculous mycobacterium, new antigens have been identified providing better opportunities for development of novel diagnostic tools [20-22]. The introduction of these antigens resulted in a much higher sensitivity and specificity in cell response assays [23]. However, serological tests based on mycobacterial antigens to detect circulating antibodies have been hampered by decreased sensitivity and cross-reactivity with other mycobacteria [24-28] or have relatively limited utility in the diagnosis of tuberculosis in countries where tuberculosis is endemic [29]. Several molecular biological techniques have been proposed as indicators of disease activity in pulmonary and extrapulmonary tuberculosis [30, 31] and are currently the most sensitive and specific diagnostic tests. However, a recent study on inter-laboratory comparisons of PCR-based TB diagnosis have demonstrated the complications of obtaining reproducible results with such sensitive techniques where false positive results can be a major problem [32].
The diagnosis of tuberculosis is currently made using one of several methods: 1) a positive culture for tuberculosis from a sputum or other biological sample, 2) a positive smear of sputum in which typical tuberculosis organisms are seen microscopically, 3) a positive histological examination of tissue from the patient, 4) a positive skin test (PPD) in a patient with a clinical examination suggestive of tuberculosis.
These methods suffer from a number of drawbacks. First, culture methods are time consuming, generally taking several weeks to perform. In addition, if the amount of sample being tested is insufficient, they result in substantial numbers of false negatives. The sputum smear suffers from poor sensitivity. The histological examination requires an invasive procedure and is generally only performed in the case of extrapulmonary tuberculosis. The skin test results in many false positive and false negative reactions; furthermore, a positive result does not distinguish between active disease, inactive infection, prior immunization with BCG vaccine, or exposure to similar organism(s).
Although a number of blood tests have been explored in attempts to overcome these problems, all have suffered from a lack of sensitivity and specificity. Thus, there is a need for a test that will more rapidly detect tuberculosis infection, and that will distinguish active disease from inactive disease, BCG vaccination, and exposure to similar organisms.
In vitro assay of antibody secretion by lymphocytes (“ALS assay”) has been previously used to measure postvaccination immunity following cholera vaccination [33] but has not been used for the detection of active infection, in particular active tuberculosis infection. In the present invention we demonstrate the diagnostic potential of tuberculosis-specific ALS responses in Bangladeshi subjects for the assessment of active pulmonary tuberculosis and detection of infection in exposed symptom-free contacts of TB index cases.